Sterile protection against malaria infection can be induced by multiple exposures to radiation-attenuated sporozoite (RAS) parasite forms in mice and humans, if the RAS remain sufficiently viable to invade hepatocytes. Manufacturing of RAS has several technical hurdles to overcome to allow mass immunization, and therefore, subunit vaccines have been the primary focus of development in recent decades. RTS,S based on Pf circumsporozoite protein (PfCSP), the predominant sporozoite surface antigen, is the most advanced subunit candidate, but has shown only limited efficacy against malaria episodes in Phase III testing. Thus, new subunit vaccine strategies are needed. CSP tolerant transgenic mice are also protected after RAS immunization, implicating additional pre-erythrocytic antigens as targets of sterile immunity. We therefore sought to identify novel candidate pre-erythrocytic vaccine antigens (PEVA) that could add to the level of protection achieved with CSP immunogens alone. We assume that PEVA candidates are transcribed during liver stage (LS) development, and have used transcriptomic data developed in our lab to identify such candidate antigens. LMIV has assessed the protective efficacy of some of these immunogens by DNA vaccination in rodent models of malaria. 1. Eight of the PEVA candidates identified from previous antigen discovery efforts using transcriptomic data are being examined side by side with five PEVA candidates identified by GenVec, a local Biotech company. All the antigens are being immunized along with CSP to examine their ability to enhance CSP-mediated protection. Both DNA prime, Ad5 boost and protein prime, Ad5 boost immunization regiments are being tested, followed by challenge with sporozoites to examine the sterile protective ability of the CSP combinations. 2. In FY 2017, the DNA prime, Ad5 boost study with the thirteen PEVA antigens mentioned above showed that four of the PEVA antigens in combination with CSP displayed higher sterile protection compared to CSP alone. 3. Eight of the PEVA antigens were also expressed and purified from either E. coli or insect cells, and were used in mice immunization studies with CSP. From the results available for four of the proteins, it could be seen that one of the proteins PySHMT had higher sterile protection compared to PyCSP alone. Interestingly, the same protein increased the protection shown by CSP in the DNA immunization study as well. Mice immunized with the second set of four proteins in combination with CSP are currently being challenged with P. yoelii sporozoites to determine whether these proteins enhance the protection shown by CSP alone. 4. To discover Pre-erythrocytic Vaccine Antigens, Phage display libraries were generated using RNA extracted from fresh and axenically cultured Plasmodium falciparum NF54 sporozoites. 5. Human Sera from protected and non-protected individuals from the Mali PfSPZ vaccine trial are currently being used to screen the libraries. Using the above two libraries and negative panning with non-protected individual sera and positive panning with sera from protected individuals, it will be possible to isolate both sporozoite-specific and liver stage-specific PE vaccine antigens. 6. Analysis of the clones obtained after negative panning with non-protected individual sera and positive panning with sera from protected individuals showed that most of the genes were either mosquito genes or were empty phages; only very few of the genes were parasite genes. Since P. falciparum genes are AT rich, these genes are not well translated by E. coli and so were less represented in the library. 7. An alternate approach needs to be undertaken to identify proteins specifically recognized by sera from protected individuals.